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Kondou et al. Journal of Biological Engineering (2019) 13:77
https://doi.org/10.1186/s13036-019-0207-y
RESEARCH Open Access
Recombinant baculovirus expressing the
FrC-OVA protein induces protectiveantitumor immunity in an
EG7-OVA mousemodel
Keigo Kondou, Tomoyuki Suzuki, Myint Oo Chang and Hiroshi
Takaku*
Abstract
Background: The baculovirus (BV) Autographa californica multiple
nuclear polyhedrosis virus has been used innumerous protein
expression systems because of its ability to infect insect cells
and serves as a useful vaccinationvector with several benefits,
such as its low clinical risks and posttranslational modification
ability. We recentlyreported that dendritic cells (DCs) infected
with BV stimulated antitumor immunity. The recombinant BV (rBV)
alsostrongly stimulated peptide-specific T-cells and antitumor
immunity. In this study, the stimulation of an immuneresponse
against EG7-OVA tumors in mice by a recombinant baculovirus-based
combination vaccine expressingfragment C-ovalbumin (FrC-OVA-BV;
rBV) was evaluated.
Results: We constructed an rBV expressing fragment C (FrC) of
tetanus toxin containing a promiscuous MHC II-binding sequence and
a p30-ovalbumin (OVA) peptide that functions in the MHC I pathway.
The results showedthat rBV activated the CD8+ T-cell-mediated
response much more efficiently than the wild-type BV
(wtBV).Experiments with EG7-OVA tumor mouse models showed that rBV
significantly decreased tumor volume andincreased survival compared
with those in the wild-type BV or FrC-OVA DNA vaccine groups. In
addition, asignificant antitumor effect of classic prophylactic or
therapeutic vaccinations was observed for rBV against
EG7-OVA-induced tumors compared with that in the controls.
Conclusion: Our findings showed that FrC-OVA-BV (rBV) induced
antitumor immunity, paving the way for its use inBV immunotherapy
against malignancies.
Keywords: Recombinant baculovirus, Wild-type baculovirus,
Fragment C of tetanus toxin, Ovalbumin, T cells, Tumorimmunity
BackgroundThe baculovirus (BV) system is used for the production
ofvarious vaccine candidates, inducing humoral and cell-me-diated
cross-immunity to viral infections [1–3]. We previ-ously
demonstrated that the wild-type (wt) BV Autographacalifornica
multiple nuclear polyhedrosis virus (AcMNPV)or BV-infected
dendritic cells (DCs) exert natural killer(NK) and CD8+ T
cell-dependent antimetastatic effects onmice, but they are CD4+ T
cell independent [4–7]. Theseantimetastatic effects involve BV
directly activating NK cells
© The Author(s). 2019 Open Access This articInternational
License (http://creativecommonsreproduction in any medium, provided
you gthe Creative Commons license, and indicate
if(http://creativecommons.org/publicdomain/ze
* Correspondence: [email protected] of Life and
Environmental Sciences, Chiba Institute ofTechnology, 2-17-1
Tsudanuma, Narashino, Chiba 275-0016, Japan
by inducing the upregulation of NK cell effector functionagainst
the tumor in a Toll-like receptor 9 (TLR9)-dependent manner [8].
Additionally, BV has been shown tosuppress liver injury and
fibrosis in vivo through the induc-tion of interferon (IFN) [9].
Molinari et al. [10] alsoreported that BV carrying ovalbumin (OVA)
on the VP39capsid protein induced antitumor immunity.On the other
hand, studies by several research groups
have demonstrated that the high titer recombinant BV(rBV)
antigen can induce specific antibodies [11–13].The high-level
transgene expression from rBV vectors iswell suited for antitumor
therapy and has been tested inanimal tumor models [14–16].
le is distributed under the terms of the Creative Commons
Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted
use, distribution, andive appropriate credit to the original
author(s) and the source, provide a link tochanges were made. The
Creative Commons Public Domain Dedication waiverro/1.0/) applies to
the data made available in this article, unless otherwise
stated.
http://crossmark.crossref.org/dialog/?doi=10.1186/s13036-019-0207-y&domain=pdfhttp://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/mailto:[email protected]
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Kondou et al. Journal of Biological Engineering (2019) 13:77
Page 2 of 8
Therefore, in the present study, an rBV-based combin-ation
vaccine was developed that expressed fragment C(FrC) of tetanus
toxin containing a promiscuous MHCII-binding sequence [17] and a
p30-OVA peptide thatfunctions in the MHC I pathway [18], and its
potentialas an antitumor vaccine was evaluated.
ResultsPreparation of BV expressing FrC-OVAThe PCR products of
OVA and FrC-DNA fragments wereinserted between the KpnI and BglII
sites under the CAGpromoter of pAc-CAG-MCS2 or pVAX1-CAG-MCS
toconstruct the recombinant plasmids FrC-OVA-pAc-CAG-MCS2 and
FrC-OVA-pVAX1-CAG-MCS, respect-ively (Fig. 1a). The insertion of
FrC-OVA into plasmidswas confirmed by RT-PCR analysis (Additional
file 1: Fig-ure S1). The production of FrC-OVA-BV (rBV) and wtBVis
described in the Materials. High-titer viruses were ob-tained,
ranging from 1 × 104 to 1 × 109 FFU/ml, and thestructure of FrC-OVA
on rBV-genomic DNA was con-firmed by western blot analysis of
rBV-infected HEK-293T cells (Fig. 1b).
Fig. 1 Construction of the BV transfer vector and FrC-OVA
expression DNA vapVAX1-CAG-MCS plasmids containing the gene
encoding the first domain ofto encode OVA 257–269 peptides fused
directly to FrC. b Expression of the OOVA-pVAX1-CAG-MCS. The cell
extracts were separated by SDS-PAGE and anawith OVA-pcDNA3.1 using
FuGEN6; lane 2, transfected with FrC-OVA-pVAX1-C
IFN-γ response in mice injected with rBVAn rBV vaccine that
expressed tetanus toxin FrC contain-ing a promiscuous MHC
II-binding sequence and a p30-OVA peptide that functions in the MHC
I pathway wasconstructed. Because FrC-OVA-BV (rBV) is specific to
theMHC I pathway, we evaluated its OVA-specific IFN-γsecretion in
vivo. The OVA-specific IFN-γ-producing T-cells from splenocytes
were analyzed using ELISPOT orCD8+ T-cell IFN-γ assays 35 days
after the intramuscularinjection of rBV, wtBV,
FrC-OVA-pVAX1-CAG-MCS orPBS on days 0 and 21 in mice (Fig. 2a). As
displayed inFig. 2b, the restimulation of rBV-immunized spleen
cellswith the OVA peptide resulted in higher levels of OVA-specific
IFN-γ compared with those in cells treated withwtBV,
FrC-OVA-pVAX1-CAG-MCS or PBS. In the rBV-immunized spleen cells
treated with the control peptideHIV-1 Gag, the level of
OVA-specific IFN-γ wasdecreased to that observed in the wtBV
control. On theother hand, as determined by the CD8+ T-cell
IFN-γassay, the rBV, wtBV and FrC-OVA-pVAX1-CAG-MCSgroups showed
higher levels of CD8+ T-cell IFN-γ thanthe PBS control group (Fig.
2c and d). These results
ccine. a Construction of the FrC-OVA-pAc-CAG-MCS2 and
FrC-OVA-the FrC of tetanus toxin (TT865–1120). These plasmids were
constructedVA protein in HEK-293 T cells infected with rBV or
transfected with FrC-lyzed by immunoblot using an anti-OVA
antibody. Lane 1, transfectedAG-MCS using FuGENE-6, lane 3,
infected with rBV at an MOI of 100
-
Fig. 2 Vaccination induces OVA-specific IFN-γ-secreting spleen
cellsor CD8+ T cells in B6 mice. a Schematic of the experimental
designof mouse immunization. Six-week-old B6 mice were vaccinated
withFrC-OVA-pVAX1-GAG-MCS, wtBV, rBV or PBS on days 0 and 21
withthe same vaccine via intramuscular injection. On day 35, the
micewere sacrificed, and their spleens were isolated. b The IFN-γ
contentsin the supernatants of spleen cells from immunized mice
weredetermined using IFN-γ ELISPOT analysis. Spleen cells were
recoveredand cultured for 24 h in the presence of OVA or HIV-1 Gag
proteins. Asa control, unstimulated spleen cells were cultured. c
Intracellularstaining of IFN-γ in splenocytes immunized with
FrC-OVA-pVAX1-GAG-MCS, wtBV, rBV or PBS as indicated above. The
spleen cells wereincubated with the OVA peptide and brefeldin A for
4 h. Theintracellular production of IFN-γ in the population of CD8+
T cells wasthen analyzed by flow cytometry. d Percentage of IFN-γ
in CD8+ Tcells. The results are representative of three independent
experimentswith six mice per group, and the error bars indicate the
standarddeviations of the mean values. *P < 0.05 (Student’s
t-test)
Kondou et al. Journal of Biological Engineering (2019) 13:77
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suggest that rBV is more efficient at activating the CD8+
T-cell-mediated response than wtBV or FrC-OVA-pVAX1-CAG-MCS
groups.
Antitumor effects of rBV against
EG7-OVA-inducedtumorsExperiments were performed to verify whether
rBV couldinduce antitumor immunity against established
subcutane-ous tumors in mice. First, a classic prophylactic
vaccinationwas examined. The experimental design is demonstrated
inFig. 3a. Mice were immunized with rBV, wtBV,
FrC-OVA-pVAX1-CAG-MCS or PBS at 35 and 14 days prior to
beinginoculated with EG7-OVA cells. The growth of the tumorsin the
rBV group was inhibited compared with that ob-served in the PBS
control group (no tumors developed),whereas the tumor growth in the
wtBV or FrC-OVA-pVAX1-CAG-MCS groups did not differ from that in
thecontrols until day 9, but inhibition was noted thereafter(Fig.
3b and c). To further determine the antitumor effectsof rBV against
EG7-OVA-induced growth, a therapeuticvaccination was performed
(Fig. 4a). The survival rates ofmice that were inoculated with
EG7-OVA cells on day 0followed by immunization with rBV, wtBV,
FrC-OVA-pVAX1-CAG-MCS or PBS on days 14 and 21 were calcu-lated.
The survival times of the mice immunized with rBVwere significantly
longer than those of the mice inoculatedwith control wtBV or
FrC-OVA-pVAX1-CAG-MCS (Fig.4b). These results indicate that
rBV-mediated protectionagainst EG7-OVA-induced tumors may be a
useful antitu-mor immunotherapy tool.
DiscussionThe viral vector vaccines established to date are
humanviral vectors that continue to give rise to problems
associ-ated with biosafety and toxicity. Furthermore,
inactivatedvaccine-mediated immunity is short-lived and
predomin-antly humoral, with poor cell-mediated immunity. Among
-
Fig. 3 Effect of a classic prophylactic vaccination with rBV on
EG7-OVA cells in mice. a Experimental dosing was used to assess
theantitumor immunity conferred by rBV. B6 mice were immunizedwith
a single intramuscular injection of FrC-OVA-pVAX1-CAG-MCS(100 μg),
wtBV (1 × 108 pfu), rBV (1 × 108 pfu) or PBS. At 14 and 35days,
EG7-OVA cells (5 × 106 cells/animal) were
administeredsubcutaneously to the immunized mice. b The tumor
volumes weremeasured every 2 days for 3 weeks. c Characterization
of theestablished EG7-OVA tumor. Similar results were obtained in
twoindependent experiments with 6 mice per group. The data
arepresented as the mean ± SD. *P < 0.05
Fig. 4 Therapeutic vaccination with rBV on EG7-OVA-inducedtumors
in mice. a Schematic of the experimental timeline. bComparison of
the antitumor immunity exhibited by rBV, FrC-OVA-pVAX1-CAG-MCS or
wtBV in B6 mice. Mice were subcutaneouslyinjected on day 0 with
EG7-OVA cells (5 × 106 cells/mouse). Survivalrates of
EG7-OVA-injected mice treated with rBV, FrC-OVA-pVAX1-CAG-MCS, wtBV
or PBS on days 14 and 21. *P < 0.01 compared withthe negative
control and wtBV groups. **P < 0.05 compared with
theFrC-OVA-pVAX1-CAG-MCS group. Similar results were obtained
fromtwo independent experiments with 6 mice per group
Kondou et al. Journal of Biological Engineering (2019) 13:77
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viral vectors, adenovirus and adeno-associated virus(AAV) have a
much lower risk of insertional mutagenesisand have been tested for
oncolytic virotherapy [19–21].Adenoviral vectors will elicit
antiviral immune responsesfollowing the first administration with a
large dose of thevector because the virus is highly immunogenic
[19–23].This reaction might preclude further use of vectors ormake
subsequent use less effective. Recently, certain non-human viral
vectors, including BV, have been explored asgene therapy vectors.
Previous studies focused on the useof BVs as vaccines in gene
therapy, as BVs do not replicatein mammalian cells and have low
cytotoxicity and favor-able biosafety features [24–28]. Kim et al.
used BV as a de-livery system to introduce telomerase reverse
transcriptase(TERT) as a potential tumor-associated antigen for
cancerimmunotherapy [29]. In immunocompetent mice, BV-TERT induced
IFN-γ T cells specific for TERT and NKcell activity in mouse
splenocytes [29]. Since surface modi-fication of the BV envelope by
the vesicular stomatitis G(VSV-G) membrane protein improves BV
transduction
in vitro or in vivo, the display of the VSV G protein(VSVG) and
heterologous peptide/protein via the GP64anchor are the most widely
adopted methods for enhan-cing the in vitro and in vivo gene
transduction efficiencyof recombinant baculoviruses [30–36]. Using
this method,LyP-1, F3, and CGKRK tumor-homing peptides were
ori-ginally identified by the in vivo screening of phage
displaylibraries [37]. The fusion proteins were successfully
incor-porated into budded virions, which showed binding abil-ities
to human breast carcinoma (MDA-MB-435) andhepatocarcinoma (HepG2)
cells that were improved two-to fivefold. These fusion proteins
inhibited virus bidingand transduction by free soluble peptides.
The solubleLyp-1 peptide induces death in cultured cancer cells
andinhibits tumor growth in mice implanted with xenografttumors.
The authors described that a BV expressing LyP-1exerted an effect
similar to that of soluble LyP-1 [38]. Inaddition to allowing
efficient BV transduction, wtBV hasbeen shown to immunostimulate
the release of inflamma-tory cytokines, including INFs, TNF-α,
IL1A, IL1B andIL6 in mammalian cells and confer protection from
lethalvirus infection in mice [39, 40]. Abe et al. reported
that
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Kondou et al. Journal of Biological Engineering (2019) 13:77
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wtBV activates proinflammatory cytokines in peritonealmacrophage
cells, splenic CD11c+ DCs, and a murinemacrophage cell line through
the TLR9/MyD88 pathwayin which the BV genome induces the innate
immune re-sponse [41]. Subsequently, our previous studies
demon-strated that BV induces the functional maturation ofhuman
monocyte-derived DCs (HCDs) and the activationof human NK cells via
BV-HCDs [6, 7]. Furthermore, weshowed that BV directly actives NK
cells via TLR9 [8].BV-DCs might therefore be a useful immunotherapy
toolfor viral infections and malignancies, particularly if used
inassociation with current virotherapies to achieve the
mosteffective results. The host innate and acquired or
adaptiveimmunity were also strongly induced by the rBV vectors.In
the present study, an anticancer combination therapy
was investigated using an rBV-based combination
vaccineexpressing the FrC of the tetanus toxin and the OVA pep-tide
(FrC-OVA), and its synergistic action as an antitumorvaccine was
evaluated. For this investigation, FrC-OVA-BV (rBV), wtBV and
FrC-OVA-pVAX1-CAG-MCS wereconstructed.Using an EG7-OVA tumor mouse
model, this study
aimed to examine whether IFN-γ production by FrC-OVA-BV (rBV)
was OVA-specific. IFN-γ release ELI-SPOT or CD8+ T-cell IFN-γ
assays were used (Fig. 2a-d).In response to the OVA peptide, rBV
exhibited signifi-cantly higher OVA-specific IFN-γ levels than wtBV
orFrC-OVA-pVAX1-CAG-MCS groups. However, com-pared with that in the
rBV-immunized spleen cells thatwere restimulated with the control
HIV-1 gag peptide, thelevel of OVA-specific IFN-γ production was
reduced tothe level observed in the wtBV control. On the other
hand,in the CD8+ T-cell IFN-γ assay, the rBV, wtBV, and
FrC-OVA-pVAX1-CAG-MCS groups displayed higher levelsof CD8+ T-cell
IFN-γ than the PBS control group. In thepresent study, the question
of whether rBV can induce theeffects of antitumor immunity against
EG7-OVA cells inmice was addressed. Classic prophylactic or
therapeuticvaccinations were examined. These vaccinations
resultedin the inhibition of tumor growth and an increased
sur-vival time in response to inoculation with rBV (Figs. 3band c,
and 4b). These results indicate that the antitumoreffects of rBV
against EG7-OVA-induced tumors are me-diated by a specific
anti-FrC-OVA immune response.
ConclusionThe results of the present study revealed that an
rBV-based combination vaccine expressing FrC-OVA suffi-ciently
induced antitumor immunity in mice with estab-lished tumors and was
more effective than wtBV. Inaddition, rBV against EG7-OVA showed a
significant anti-tumor effect in classic prophylactic or
therapeutic vaccina-tions. Furthermore, baculovirus has several
attractiveadvantages, such as its good biosafety, large capacity
for
foreign genes, and better posttranslational modificationsthan
those of other gene delivery vehicles. Therefore, rBVis potentially
useful as an efficient antimetastatic agentand is expected to be
advantageous in the future develop-ment of antitumor therapies.
MaterialsCell culture and reagentsFemale C57BL/6 mice (B6) (6
weeks old) were purchasedfrom Japan SLC, Inc. and maintained under
humane andspecific pathogen-free conditions according to the
rulesand regulations of the institutional committee of
ChibaInstitute of Technology, Narashino, Japan.
Spodopterafrugiperda (Sf9) insect cells were cultured in Sf-900
IIculture medium (Invitrogen; Thermo Fisher Scientific,Inc.).
HEK-293 T cells were cultured in DMEM (Sigma-Aldrich; Merck KGaA)
supplemented with 10% fetal bo-vine serum (FBS; Thermo Fisher
Scientific, Inc.), 100 U/ml penicillin and 100 μg/ml streptomycin
(both Sigma-Aldrich; Merck KGaA). EG7-OVA cells (EL4
derivative,ATCC® CRL-2113™, American Type Culture Collection)were
maintained in complete RPMI 1640 medium(Gibco; Thermo Fisher
Scientific, Inc.) supplementedwith 10% heat-inactivated fetal calf
serum, 2 mML-glutamine, penicillin (0.1 U/ml) and streptomycin
(0.1mg/ml) and adjusted to contain 1.5 g/l sodium bicarbon-ate, 4.5
g/l glucose, 10 mM Hepes, 1 mM sodium pyru-vate, 0.05 mM
2-mercaptoethanol and 0.4 mg/ml G418at 37 °C in a 5% CO2
atmosphere.
PlasmidsThe DNA vaccine containing the gene encoding the
firstdomain (p. DOM) was constructed by PCR amplificationof the
N-terminal domain sequence (TT865–1120) fromp. FrC using the
forward primer Kpn I-DOM-F
(5′-CGGGGTACCGCCGCCACCATGGGTTGGAGCTGTATCAT-3′) and the reverse
primer Bgl II-DOM-R contain-ing the OVA peptide sequence (257–269)
(5′-GAAGATCTTTAACTGGTCCATTCAGTCAGTTTTTCAAAGTTGATTATACTGTTACCCCAGAAGTCACGCA-3′)
be-fore cloning into pAc-CAG-MCS2. To generate pVAX1-CAG-MCS, the
MluI/BamHI-digested DNA fragment ofpAc-CAG-MCS2 [42] was inserted
into MluI/BamHI-digested pVAX1-CMV-MCS. The PCR product (FrC-OVA)
was inserted between the Kpn I and Bgl II sitesunder the CAG
promoter of pAc-CAG-MCS2 or pVAX1-CAG-MCS. The PCR products of OVA
and the Fc-DNAfragments were cloned into pcDNA3.1 to construct the
re-combinant OVA-pcDNA3.1 plasmids. The plasmids prop-agated in
Escherichia coli were purified with the QiagenPlasmid Mini kit
(Qiagen GmbH) according to the manu-facturer’s protocols. The
insertion of FrC-OVA into plas-mids was confirmed by RT-PCR
analysis of total RNA
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Kondou et al. Journal of Biological Engineering (2019) 13:77
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isolated from rBV-infected HEK-293 T cells (Additionalfile 1:
Figure S1) and sequence analysis.
Preparation of BVAcMNPV and rBV were propagated in Sf9 cells
culturedin TMN-FH medium (BD Biosciences) containing100 μg/ml
kanamycin and 10% FBS. Ac/CAG-FrC-OVAand AcMNPV were purified as
described previously [39,43, 44]. The viral titers were determined
by a plaqueassay.
Reverse transcription (RT)-PCR analysisTotal RNA was extracted
from cells using the GenElute™Mammalian Total RNA Miniprep kit
(Sigma-Aldrich;Merck KGaA) according to the manufacturer’s
proto-cols. cDNA was prepared using ReverTra Ace-α-™(Toyobo Life
Science). The PCRs for FrC-OVA andGAPDH were performed using TaKaRa
Ex Taq™, Hot-Start Version (Takara Bio, Inc). The primer
sequenceswere as follows: GAPDH forward, 5′-GGTGAAGGTCGGTGGAACG-3′
and reverse, 5′-CTCGCTCCTGGAAGATGGTG-3′. The PCR conditions
consisted of aninitial denaturation step at 94 °C for 3 min,
followed by30 cycles of denaturation at 94 °C for 30 s, annealing
at64 °C for 30 s, and extension at 72 °C for 12 s.
TransfectionsHEK-293 T cells (3 × 105 cells/well in 24-well
plates)were transfected with 1.0 μg of the FrC-OVA-pAc-CAG-MCS2 and
FrC-OVA-pVAX1-CAG-MCS plasmids usingFuGENE-6 (Roche Diagnostics).
At 20 h post transfec-tion, RT-PCR was used to analyze the
insertion of theFrC-OVA gene into the FrC-OVA-pAc-CAG-MCS2
andFrC-OVA-pVAX1-CAG-MCS plasmids. The details ofthe FrC-OVA RNA
analysis are described in the previ-ous section.
Detection of the OVA protein in virus-infected ortransfected
cells by western blot analysisHEK-293 T cells were infected at an
MOI of 100 with rBV(1 × 108 pfu) or transfected with
FrC-OVA-pVAX1-CAG-MCS (1.0 μg) using FuGENE-6. At 24 h post
infection, thecells were lysed with lysis buffer (50mM Tris-HCl, pH
6.8,0.1M dithiothreitol, 2% SDS and 10% glycerol). The cellextracts
were separated by SDS-PAGE, and proteins wereblotted onto a PVDF
membrane (Roche MolecularDiagnostics). The OVA protein was detected
using ananti-OVA antibody (cat. no. SAB5300165; 1:2000
dilution;Sigma-Aldrich; Merck KGaA) and a
horseradishperoxidase-conjugated anti-mouse IgG secondary
antibody(1:10,000 dilution) using an ECL plus detection system(both
GE Healthcare). GAPDH was used as an internalcontrol.
In vivo IFN-γ ELISPOT and CD8+ T-cell IFN-γ
assaysFrC-OVA-pVAX1-CAG-MCS (100 μg), wtBV (1 × 108
pfu), rBV (FrC-OVA-BV; 1 × 108 pfu) or PBS was adminis-tered to
the mice via intramuscular injection, and the in-jections were
repeated on day 21. On day 35 postinjection, the mice were
sacrificed, and their spleens wereisolated. Dissociation of the
mouse spleens was performedusing the gentleMACS Dissociator
(Miltenyi BiotecGmbH) according to the manufacturer’s protocol.
Follow-ing centrifugation (1500×g, 5 min, room temperature),
thesupernatant fluid was removed, and the pellet was resus-pended
in culture medium at the desired concentration.An aliquot of the
suspended cells was used to assay thecell quantity, and the cell
suspension was adjusted to afinal density of 1xl07 cells/ml. The
splenocytes were usedfor the IFN-γ ELISPOT and CD8+ T-cell IFN-γ
assays.IFN-γ release was measured using a commercial mouseIFN-γ
ELISPOT Ready SET Go kit (eBioscience; ThermoFisher Scientific,
Inc.) according to the manufacturer’s in-structions. Mouse spleen
cells (2 × 106 cells/well) wereadded to 96-well PVDF plates (Merck
KGaA) that wereprecoated with a capture mouse anti-IFN-γ
monoclonalantibody (5 μg/ml). The plates were treated with the
OVA257–264 peptide (GenScript Inc.) or HIV-1 Gag (#11057;129
peptide consensus group M sequences, AIDS Re-search and Reference
Reagent Program, Division of AIDS,National Institute of Allergy and
Infectious Diseases, Na-tional Institutes of Health) at a
concentration of 2.0 μg/well. After inoculation for 24 h at 37 °C,
the plates werewashed three times with PBS-Tween 0.05% and
incubatedwith a biotinylated anti-IFN-γ monoclonal antibody at100
μg/well for 2 h at room temperature. Following an-other round of
washing, the plates were incubated withstreptavidin-conjugated
alkaline phosphatase for 1 h atroom temperature. After the plates
were washed with ELI-SPOT wash buffer, each well was incubated with
AECsubstrate solution (100 μl/well) for 24 h at 37 °C. The platewas
air-dried overnight at room temperature in the dark.The ELISPOT
plate was read by ZellNet Consulting, Inc.The CD8+ T-cell IFN-γ
assays were carried out using a
commercial BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization
kit (BD Biosciences) according to the man-ufacturer’s protocols.
The mouse spleen cells (2 × 106 cells/well) were incubated with the
OVA 257–264 peptide(2.0 μg/1.0ml) and brefeldin A (1 μg/1.0ml) for
4 h at 37 °C.Subsequently, the mouse spleen cells were incubated
with aCD16/D32 monoclonal antibody (eBioscience; ThermoFisher
Scientific, Inc.) for 15min on ice in the presence of a2.4G2
monoclonal antibody to block FcγR binding. Follow-ing blocking, the
cells were treated with FACS buffer(900 μl) and centrifuged at
2000×g for 5min at 4 °C. The re-suspended cells were treated with
fixation/permeabilizationsolution (100 μl) for 20min at 4 °C and
then washed twicewith BD Perm/Wash™ buffer. The cells were
incubated with
-
Kondou et al. Journal of Biological Engineering (2019) 13:77
Page 7 of 8
FITC-anti-mouse IFN-γ (10 μl; eBioscience; Thermo
FisherScientific, Inc.) at 4 °C for 30min in the dark and
thenwashed twice with 1.0ml BD Perm/Wash buffer. The plateswere
incubated with PE-conjugated anti-mouse CD8a (BDBiosciences) at 4
°C for 30min in the dark and washed twicewith 1.0ml of FACS buffer.
The cell pellets were suspendedin FACS buffer (500 μl) and analyzed
on a FACSCalibur in-strument with CellQuest Pro software (BD
Biosciences).
In vivo immunization therapy against EG7-OVA cellsFor
prophylactic assessment, C57BL/6NCrSlc mice wereimmunized by a
single intramuscular injection of FrC-OVA-pVAX1-CAG-MCS (100 μg),
wtBV (1 × 108 pfu),rBV (1 × 108 pfu) or PBS. After 14 and 35 days,
EG7-OVA cells (5 × 106 cells/mouse) were transplanted
sub-cutaneously into the immunized mice. The tumor vol-ume was
measured every 2 days for 3 weeks using aslide caliper and
calculated according to the followingformula: tumor volume (mm3) =
0.5 × length (mm) ×width2 (mm2). To assess the therapeutic effect,
C57BL/6NCrSlc mice were subcutaneously injected on day 0with
EG7-OVA cells (5 × 106 cells/mouse). The survivalrates of
EG7-OVA-injected mice treated with rBV (1 ×108 pfu), wtBV (1 × 108
pfu), FrC-OVA-pVAX1-CAG-MCS (100 μg) or PBS (days 14 and 21) were
evaluated.
Statistical analysisOne-way analysis of variance followed by
Tukey’s posthoc test or the Mann-Whitney U test were conductedfor
pairwise comparisons. All calculations were per-formed using the
Statistica program (StatSoft, Inc.). Theresults are presented as
median or mean values ± stand-ard deviations. P < 0.05 was
considered to indicate a sta-tistically significant difference.
Supplementary informationSupplementary information accompanies
this paper at https://doi.org/10.1186/s13036-019-0207-y.
Additional file 1: Figure S1. RT-PCR analysis FrC-OVA RNA
expressionin Frc-OVA-pAc-CAG-MCS2 and FrC-OVA-pVAX1-CAG-MCS. (a, c)
Sche-matic maps of Frc-OVA-pAc-CAG-MCS2 and
FrC-OVA-pVAX1-CAG-MCSplasmids. RT-PCR amplification products
analyzed by 2% agarose gel elec-trophoresis with ethidium bromide
staining. RT-PCR analysis of FrC-OVARNA was carried out using
FrC-OVA specific primers with concomitantamplification of GAPDH
mRNA. Lane 1: MOCK; lane 2: PC; lane 3: FrC-OVARNA expression in
FrC-OVA-pAc-CAG-MCS2 (b) and FrC-OVA-pVAX1-CAG-MCS (d).
AbbreviationsAcMNPV (wtBV): Autographa californica multiple
nuclear polyhedrosis virus;CD8+T cells: CD8-positive T-lymphocyte;
CMV promoter: Cytomegaloviruspromoter; EG7-OVA cells:
OVA-expressing EG7 lymphoma cells;ELISPOT: Enzyme-linked
immunospot; FACS: Fluorescence-activated cellsorting; FrC: Fragment
C; HEK-293 T cells: Human embryonic kidney cells 293;MHC: Major
histocompatibility complex; OVA: Ovalbumin; rBV: RecombinantBV; Sf9
cells: Spodoptera frugiperda (Sf9) insect cells
AcknowledgementsThe authors thank A. Fujihira and T. Moriyama
for their excellent technicalassistance.
Authors’ contributionsHT and TS conceived and designed the
experiments. KK, TS and HTperformed the experiments. TS, KK, MOC
and HT analyzed the data. HT andTS wrote the paper. All authors
have read and approved the finalmanuscript.
FundingThis work was supported in part by a grant from the
Supporting Program forCreating University Ventures from the Japan
Science and TechnologyAgency and by a grant for Research and a
Grants-in-Aid for AIDS researchfrom the Ministry of Health, Labor
and Welfare, Japan.
Availability of data and materialsThe datasets used and/or
analyzed in the current study are available fromthe corresponding
author upon reasonable request.
Ethics approval and consent to participateAll study protocols
were approved by the Animal Welfare Committee ofChiba Institute of
Technology, Narashino, Japan.
Consent for publicationNot applicable.
Competing interestsThe authors declare that they have no
competing interests.
Received: 5 June 2019 Accepted: 16 September 2019
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Publisher’s NoteSpringer Nature remains neutral with regard to
jurisdictional claims inpublished maps and institutional
affiliations.
AbstractBackgroundResultsConclusion
BackgroundResultsPreparation of BV expressing FrC-OVAIFN-γ
response in mice injected with rBVAntitumor effects of rBV against
EG7-OVA-induced tumors
DiscussionConclusionMaterialsCell culture and
reagentsPlasmidsPreparation of BVReverse transcription (RT)-PCR
analysisTransfectionsDetection of the OVA protein in virus-infected
or transfected cells by western blot analysisIn vivo IFN-γ ELISPOT
and CD8+ T-cell IFN-γ assaysIn vivo immunization therapy against
EG7-OVA cellsStatistical analysis
Supplementary informationAbbreviationsAcknowledgementsAuthors’
contributionsFundingAvailability of data and materialsEthics
approval and consent to participateConsent for publicationCompeting
interestsReferencesPublisher’s Note